Pharmaceutical compositions and use thereof in treating inflammation

ABSTRACT

Inflammation in mammals is treated by administering anti-inflammatory agent complexed with corticosteroid binding globulin (CBG).

This application is a division, of application Ser. No. 07/204,356,filed June 9, 1988, now U.S. Pat. No. 4,997,814.

FIELD OF THE INVENTION

This invention relates to anti-inflammatory agents such asglucocorticoids and their use in treating inflammation.

BACKGROUND TO THE INVENTION

Glucocorticoids are steroid hormones, many of which are potentanti-inflammatory agents. Their physiological effects are not limited totheir anti-inflammatory properties, however, nor are their effectsrestricted specifically to inflamed tissue. Protein, lipid andcarbohydrate metabolism can be altered adversely and electrolyte balancecan be disturbed particularly when large or repeated therapeutic dosesof the anti-inflammatory glucocorticoids are administered.

Certain recently developed synthetic glucocorticoids show promise astherapeutic alternatives to the natural anti-inflammatoryglucocorticoids such as cortisone, cortisol and corticosterone. Thesynthetic analogues, which include dexamethasone and betamethasone,exhibit reduced effects on electrolyte balance and tend therefore toelicit fewer adverse side effects. In many instances, they also exhibitgreater potency as anti-inflammatory agents relative to their naturalcounterparts, presumably owing to their reduced binding affinity for theplasma protein known as corticosteroid binding globulin (CBG). It hasbeen reported that natural glucocorticoids become biologicallyinactivated upon binding with CBG in the circulation whereas manysynthetic glucocorticoids do not bind and thus remain active (seeMickelson et al., Biochemistry 1981, 20, 6211-6218 where bindingaffinities for some natural and synthetic glucocorticoids relative toCBG are tabled). The capacity of synthetic glucocorticoids to escapeCBG-binding and thus to remain free and biologically active followingadministration is presumed to be at least partly responsible for theirenhanced anti-inflammatory properties in vivo.

Apart from the numerous physiological effects exerted byglucocorticoids, their use in treating inflammation is furthercomplicated by their ability to affect a broad range of tissues, whetherinflamed or healthy. Because so many tissues are responsive tocorticosteroids, they are rarely administered systemically unlessinflammation is particularly severe or life threatening.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a strategyuseful therapeutically for localizing the effects of anti-inflammatoryagents, such as the glucocorticoids, predominantly to a site ofinflammation.

In accomplishing this object, there is provided, in accordance with oneaspect of the invention, a pharmaceutical composition useful in treatinginflammation in a mammal which comprises CBG, an anti-inflammatory agentwhich binds therewith, and a pharmaceutically acceptable carrier. Anyanti-inflammatory agent which binds with appropriate affinity to CBG maybe used in the present composition. Preferably, the anti-inflammatoryagent is present in an amount sufficient to bind substantially all ofthe CBG present in the composition. In one preferred embodiment of theinvention, the anti-inflammatory agent is a glucocorticoid. Generally,the amount of anti-inflammatory agent required in the presentcomposition is less than that required in the absence of CBG since, aswill be explained, CBG is capable of localizing the effects of theanti-inflammatory agent to an inflammation site.

In accordance with another aspect of the invention, there is provided amethod for treating inflammation in a mammal which comprisesadministering thereto a pharmaceutical composition comprising CBG-boundanti-inflammatory agent.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that the binding affinity of CBG for suchanti-inflammatory agents as the glucocorticoids can be exploited toimprove upon their therapeutic effectiveness as anti-inflammatory agentseven though such agents are rendered biologically inactive when bound byCBG. More particularly, it has been found that CBG-bound glucocorticoidscan be released from CBG at local sites of inflammation by the action ofleukocyte elastase, an enzyme which is prevalent at sites ofinflammation. Leukocyte elastase specifically cleaves the CBG componentof the glucocorticoid: CBG complex, allowing glucocorticoid todissociate from CBG and exert its anti-inflammatory action directly atthe inflammation site. Accordingly, administration of glucocorticoid inCBG-bound form is an effective means for localizing glucocorticoideffects to those tissues or sites at which inflammation is active, thusreducing the indiscriminate stimulation of other tissues byglucocorticoids which results in undesirable side effects. Similarly,any anti-inflammatory agent which binds with CBG can be targetted forrelease specifically at an inflammation site by administering thatparticular agent in CBG-bound form.

That CBG is cleaved specifically by leukocyte elastase, a serineprotease released locally by neutrophils at sites of inflammation, isparticularly remarkable. First, the region that contains the proposedelastase binding site on another elastase substrate, α₁ -antitrypsin, isnot well conserved when compared with CBG. Moreover, again by analogywith α₁ -antitrypsin, the putative elastase cleavage site on CBGinvolves a neighboring glycosylation site which is utilized in the CBGmolecule, and is not involved in α₁ -antitrypsin. Yet, the experimentaldata herein provided demonstrates clearly that CBG is indeed cleaved byleucocyte elastase, at a site which is very close to that on theoxidized α₁ -antitrypsin molecule which is cleaved by macrophageelastase, and shows also that when cleaved by elastase, CBG losessteroid binding activity.

In accordance with the invention, anti-inflammatory agents which bindwith CBG are administered in CBG-bound form to treat inflammation inmammals. It should be appreciated that the binding of a particularanti-inflammatory and CBG is mediated by the affinity of one moleculefor the other and not through a physical link such as a convalent bondor chemical linking agent. The degree of affinity between CBG and theselected anti-inflammatory agent should be sufficient to enable CBG andthe agent to remain in association following administration thereof tothe mammal to be treated, and allow the corticosteroid to dissociatefollowing elastase-induced cleavage of CBG at an inflammation site. Toidentify those anti-inflammatory agents suitable for use in the presentinvention, one simply determines whether CBG is capable of binding witha selected anti-inflammatory agent. In general, an anti-inflammatoryagent having an affinity constant for CBG within the range from 0.1 to10 nanomolar at about 4° C. and physiological pH is suitable for usewith CBG according to the present invention. A protocol for screeninganti-inflammatory agents for suitability in the present context isdescribed by Mickelson et al., supra, incorporated herein by reference,which allows the binding affinity of a selected steroid for CBG to becompared with the affinity of CBG for one of its natural ligands,cortisol. Any anti-inflammatory agent having CBG binding affinity whichapproximates that of cortisol is suitable for use in accordance with thepresent invention. Mickelson et al., supra, also identify some criteriauseful in predicting whether a particular steroid will be capable ofbinding adequately with CBG such as the presence of a 3-oxo group andthe absence of an 11 α-hydroxy group on the steriod. While thesecriteria can be useful in determining binding affinity of steroid forCBG, it should be understood that the determinative measure ofsuitability of anti-inflammatory agent, whether steroidal in structureor not, is stability of the agent in CBG-bound form under the conditionsnoted above.

Specific anti-inflammatory agents useful herein include corticosteroidshaving glucocorticoid activity such as cortisol and other naturalcorticosteroids exhibiting anti-inflammatory properties such ascorticosterone and cortisone. Synthetic glucocorticoids which exhibitreduced adverse effects on electrolyte balance, such as prednisolone andmethylprednisolone are particularly suitable although, by intentionaldesign, most other of the currently available synthetic glucocorticoidshave an inappropriately low binding affinity for CBG. Nevertheless,synthetic corticosteroids developed subsequently or naturalcorticosteroids discovered hereafter are useful provided, of course,that they exhibit the appropriate level of anti-inflammatory activityand an appropriate CBG binding affinity, preferably a binding affinityfor CBG which approximates that of cortisol (about 0.7 nanomolar at 4°C., physiological pH, as measured using the protocol described byMickelson et al., supra).

The anti-inflammatory agent used in the pharmaceutical compositions ofthe invention should be of pharmaceutical grade. Whereas acid- andbase-addition glucocorticoid salts are commonly employed in prior artcompositions to enhance solubility of the glucocorticoid, in injectablesolutions for example, salt forms of the selected glucocorticoid are notrequired in the present compositions although they may be used, ifdesired. When complexed with CBG, glucocorticoids are sufficientlysoluble in solution in the amounts useful to treat inflammation,although solubilizers standard in the protein formulation art, such asmannitol, may be used.

CBG useful in the composition of the invention is now a wellcharacterized protein, having been identified in human plasma overthirty years ago. It has since been identified in virtually everyvertebrate species examined and has been isolated from numerousmammalian species including rabbit, guinea pig, rodent, humans andothers. Techniques for isolating CBG from serum are now well known inthe art. The most practical and efficient of these techniques involvethe use of affinity columns in which the immobilized ligand is either aglucocorticoid or a derivative thereof which binds with high affinity toCBG. The preparation of such a column in which a cortisol derivative iscross-linked on Sepharose and its use in isolating CBG are described byRosner in J. Steroid Biochem, 1972, 3, 531-542. An improved method forpurifying human CBG from pregnancy serum, in which CBG levels are about2-fold higher than normal serum, is described by Robinson et al in J.Endocr. (1985) 104, 259-267, incorporated herein by reference. Theprocedure entails applying serum to a column in which the ligand is acorticosterone derivative, collecting the CBG-containing eluate and thensubjecting the eluate to successive purification steps such as gelelectrophoresis and including a step in which contaminating albumin isremoved.

Antibodies to CBG have also now been prepared (see Robinson et al.,supra) and may also be employed to isolate CBG from serum.

In general, provided that the CBG used in the composition has theappropriate binding affinity for the selected glucocorticoid and issusceptible to cleavage by elastase, such CBG is suitable. It isconceivable that elastase endogenous in the mammalian species to betreated may have some specificity for the CBG native to that species.Given the importance of CBG cleavage by elastase in the present context,it is recommended that the administered composition comprises CBG nativeto the mammalian species being treated. For example, human CBG ispreferably used in compositions administered to treat inflammation inhumans and equine CBG is used when horses are to be treated.

To prepare compositions of the invention, the selected anti-inflammatoryagent and CBG are simply admixed in solution, such as physiologicalsaline, at pH 7.5, with agitation to generate the required complexes.Formation of CBG:anti-inflammatory agent complexes can be facilitated byincubating at physiological temperature and pH. It is important, whenCBG-bound glucocorticoids are prepared to maintain pH above 6 becausethe affinity of CBG for glucocorticoid is reduced below pH 6. It isdesirable, in order to achieve the maximum localizing effect of CBG,that the CBG in the administered composition is saturated withglucocorticoid i.e. the molar ratio of glucocorticoid bound to CBG inthe composition is substantially 1:1. To accomplish this, it ispreferred to use a molar excess of glucocorticoid such as a molar ratioof 2:1 (glucocorticoid:CBG) when generating the complexed material.Unbound glucocorticoid may remain with the complexed material, ifdesired.

To formulate CBG-bound glucocorticoid for administration, any of theconventional pharmaceutically acceptable carriers may be used, selectionof which depends on the intended mode of administration and is dictatedto some extent by the proteinaceous nature of the complexes. Injectablesolutions may be prepared using well known liquid vehicles such asbuffered saline and physiological saline. Compositions for topicalapplication may be prepared as creams, lotions or ointments using anappropriate base such as triglycerides. Surface active agents may alsobe used as may preservatives to prevent microbial growth over prolongedstorage periods. Aerosol inhalable formulations of CBG-boundglucocorticoid may also be prepared using propellant and an appropriateliquid vehicle.

The compositions of the invention are useful in pharmaceutical and inveterinary applications to treat a variety of conditions involvinginflammation. It will be appreciated that the compositions areparticularly better adapted for systemic administration than knowncompositions comprising free anti-inflammatory agent since CBG-bindingthereof is capable, by the limiting of therapeutic action to aninflammation site, of reducing the potential for adverse side effectselsewhere in the body. Because of the effect which CBG has oncontrolling release of glucocorticoid, for example, it may be used inthe present compositions in lower amounts relative to compositions ofglucocorticoid per se, to elicit an anti-inflammatory response. Whenadministered systemically as an injectable solution, formulations willcomprise glucocorticoid (as CBG-bound glucocorticoid) in a molar rangeof 5-500 μM. To determine appropriate unit doses for treating a specificmedical conditions with a particular anti-inflammatory agent, referencemay be made to product monographs or more generally to Pharmacopoeias,such as Martindale, The Extra Pharmacopoeia Ed, J. E. F. Reynolds, ThePharmaceutical Press, London, 1982 (see particularly pp 446-485).

The compositions of the invention are useful to treat the sameinflammation-related medical conditions or disorders as are treatable bythe anti-inflammatory agents per se. In this connection, reference maybe made to The Extra Pharmacopoeia cited above and incorporated hereinby reference. For example, administration systemically of CBG-boundanti-inflammatory agent will be useful to treat blood disordersincluding auto-immune haemolytic anemia, idiopathic thrombocytopenicpurpura, arthritis, septicemia etc. Local injection may be appropriateto treat collagen and rheumatoid disorders as well as certain connectivetissue disorders. In cream, ointment or lotion form, the compositionsmay be applied to treat inflammation active at exposed areas of theskin. Topical administration of the compositions may be applied to openwounds to reduce inflammation. The administration of aerosols isappropriate for treating adult respiratory distress syndrome,respiratory ailments such as steroid dependent asthma and interstitallung disease. In general, administration of the compositions of theinvention will be useful to reduce inflammation in areas where theCBG-corticosteroid complexes can become exposed, followingadministration, to the action of elastase.

Demonstration that Leucocyte Elastase Cleaves CBG

Human CBG was purified by affinity chromatography and Blue Sepharosechromatography, as previously described (Robinson et al., (1985) J.Endocrinol. 104:250). To evaluate the relationship of CBG to serineproteases, approximately lug pure CBG was incubated (5 min at 37° C.) in30 mM Tris, pH 7.5 (or pH optimal appropriate for the different serineproteases), with 20 ng human leucocyte elastase (Elastin ProductsCompany, Inc), cathepsin G (EPC), thrombin (Boehringer Mannheim Canada)or plasmin (BMC). The reaction was terminated by boiling in 5 μlSDS-PAGE loading buffer, and then subjected to electrophoresis on a12%-SDS polyacrylamide gel. After the gel was stained with CoomasieBlue, it was apparent that, under the experimental conditions, a clear˜5 kDa reduction in the apparent M_(r) of human CBG occurred only afterincubation with elastase.

In order to determine the site of cleavage, CBG and elastase were againincubated under the same conditions. The reaction was "snap frozen" andstored at -70° C. until subjected to amino-terminal sequence analysis.The results revealed two CBG fragments: the authentic amino-terminus ofmature human CBG, (Ferlund and Laurell (1981) J. Steroid Biochem.14:545), together with a second sequence which was identified asstarting with residue 342 (Thr) in the mature form of human CBG (Hammondet al. (1987) Proc. Natl. Acad. Sci. USA 84:5153).

The steroid binding properties of human CBG were also tested using acortisol binding capacity assay (Hammond and Lahteenmaki (1982) Clin.Chem. Acta 132:101) before and after a standard incubation withelastase, as described above. In this way it could be shown that >95% ofthe steroid binding activity of human CBG was lost after treatment withelastase, indicating that CBG had lost its binding affinity for theanti-inflammatory agent cortisol following exposure to elastase.

I claim:
 1. A pharmaceutical composition useful in treating a mammalsuffering from inflammation, said composition comprising human CBG, ananti-inflammatory agent which binds therewith, and a pharmaceuticallyacceptable carrier, wherein the said anti-inflammatory agent has anaffinity constant for CBG within the range from 0.1 to 10 nanomolar atabout 4° C. and physiological pH.
 2. The composition according to claim1, wherein said anti-inflammatory agent is a glucocorticoid.
 3. Thecomposition according to claim 2, wherein the anti-inflammatory agent iscortisol.
 4. A composition according to claim 1 which is an injectablesolution.
 5. A composition according to claim 1 in a form suitable fortopical administration.
 6. A composition according to claim 1 which isan aerosol.
 7. A pharmaceutical composition useful in treating a mammalsuffering from inflammation, said composition comprising a CBG, ananti-inflammatory agent which binds therewith, and a pharmaceuticallyacceptable carrier selected from a composition for topical applicationand an aerosol inhalable formulation.
 8. The composition according toclaim 7, wherein the CBG is human CBG.
 9. The composition according toclaim 7, wherein said anti-inflammatory agent is a glucocorticoid. 10.The composition according to claim 9, wherein the anti-inflammatoryagent is cortisol, wherein the said anti-inflammatory agent has anaffinity constant for CBG within the range from 0.1 to 10 nanomolar atabout 4° C. and physiological pH.
 11. The composition according to claim7 in a form suitable for topical administration.
 12. The compositionaccording to claim 7 which is an aerosol.
 13. A method for treating amammal suffering from inflammation, comprising the step of administeringto said mammal a pharmaceutical composition comprising CBG, ananti-inflammatory agent which binds therewith, and a pharmaceuticallyacceptable carrier.
 14. The method according to claim 13, wherein saidanti-inflammatory agent is a glucocorticoid.
 15. The method according toclaim 14, wherein said anti-inflammatory agent is cortisol.
 16. Themethod according to claim 13, wherein said composition is administeredby injection, wherein the said anti-inflammatory agent has an affinityconstant for CBG within the range from 0.1 to 10 nanomolar at about 4°C. and physiological pH.
 17. The method according to claim 13, whereinsaid composition is administered topically.
 18. The method according toclaim 13, wherein said composition is administered by inhalation. 19.The method according to claim 13, wherein the CBG is human CBG.
 20. Themethod according to claim 19, wherein said anti-inflammatory agent is aglucocorticoid.
 21. The method according to claim 20, wherein saidanti-inflammatory agent is cortisol.
 22. The method according to claim19, wherein said composition is administered by injection.
 23. Themethod according to claim 19, wherein said composition is administeredtopically.
 24. The method according to claim 19, wherein saidcomposition is administered by inhalation.